Polypeptides from CREB binding protein and related protein p300 for use in transcriptional regulation

ABSTRACT

A method for determining whether a compound inhibits or disrupts an interaction between a first polypeptide comprising a transcriptional adaptor motif (TRAM) and a second polypeptide comprising a TRAM-interaction motif. The first polypeptide and/or second polypeptide may be Mdm-2, p53, TBP, E2F, YY1, CBP/p300 or TF11B, or a viral polypeptide such as a human papillomavirus (HPV) E6 polypeptide from HPV strain (16) or (18).

FIELD OF THE INVENTION

The present invention relates to polypeptides which compriseTRanscriptional Adaptor Motif (TRAM) and/or TRAM-Interaction Motif(TRIM) sequences. These polypeptides may be used in assay methods toidentify inhibitors of TRAM/TRIM interactions. They may also be used inmethods of treating viral disease or cancer where a TRAM/TRIMinteraction is important in disease initiation/progression.

BACKGROUND TO THE INVENTION

The control of the cell cycle, during which cells replicate their DNAand divide, is a cardinal step in normal human cell growth and in tissuedifferentiation, while de-regulation of this process can result intumourigenesis and the onset of cancer. Protein-protein interactionsplay a crucial role in these events, with the regulation of geneexpression in particular being central for the determination of cellcycle fate.

Recently, the transcriptional co-factor CBP (CREB binding protein) andthe related protein p300 have been implicated in the control of cellcycle events. Interestingly, CBP has been shown to be required for bothE2F activity which results in the expression of S phase specific genesand cellular proliferation, and for the expression of p21^(WAF) by p53,which results in cell cycle arrest. Moreover, studies of cellulartransformation by the adenovirus oncogene product E1A have shown that aninteraction with CBP is necessary for this process. These observationssuggest that two opposite cell cycle events rely upon an interactionwith a common factor, CBP/p300.

CBP is a very large protein (2,441 amino acids) and can be thought of asa transcriptional adaptor with the capability of binding many differenttranscriptional factors. While a few regions of CBP have been describeda “hot spots” for protein interactions, the mechanisms by whichdifferent proteins interact with CBP, and the exact motifs involved,have not been defined. Many of the proteins that regulate the cell cyclebind to the same 257 amino acid region of CBP (1621-1877). However,since a number of these proteins are thought to have antagonist effects,it would be useful to know if the same CBP sequences were recognized bythese different proteins or if not, if the different motifs overlapped.

SUMMARY OF THE INVENTION

We have now identified a TRAM within CBP and also in the related p300.These TRAMs are conserved between species. They provide a consensus TRAMthat has been shown to bind to multiple cellular regulators includingthe cellular transcription factors p53, E2F, TFIIB and YYI. However,TRAMs are not limited to CBP/p300 since one is also found in Mdm-2proteins.

TRAM-interacting proteins may bind a TRAM sequence through differentTRIMs. A TRIM therefore binds a TRAM sequence, typically a TRAM sequenceof CBP or p300 or Mdm-2. One type of TRIM may be defined by theconsensus sequence FXE/DXXXL. The variation seen in potential TRIMs ledus to check a series of TRAM mutants (alanine-substitutions) againstdifferent TRAM-interacting proteins. We found differential bindingproperties of TRAM variants with respect to TRAM-interacting proteins.Thus TRAM variants can be used to add specificity TRIM-TRAMinteractions.

Since the TRIM/TRAM-containing proteins identified here are involved inthe processes of transcriptional regulation, cell cycle control andviral infection, the identification of compounds which disruptinteractions between, for example cellular TRAM-containing proteins andcellular TRIM-containing proteins, or cellular TRIM/TRAM containingproteins and viral TRIM/TRAM proteins may allow these processes to betargeted in the treatment of, for example, tumours and viral diseases.

Accordingly the present invention provides a method for determiningwhether a compound is capable of inhibiting or disrupting an interactionbetween a first polypeptide and a second polypeptide said methodcomprising:

-   -   (a) (i) incubating said first polypeptide with said second        polypeptide under conditions which allow the first polypeptide        to bind to the second polypeptide to form a complex; and        bringing the complex thus formed into contact with a candidate        compound; or        -   (ii) incubating said first polypeptide with said second            polypeptide in the presence of a candidate compound under            conditions which would allow the first polypeptide to bind            to the second polypeptide in the absence of the candidate            compound; and    -   (b) determining if said candidate compound inhibits or disrupts        binding of the first polypeptide to the second polypeptide;        wherein said first polypeptide comprises a TRAM sequence and        said second polypeptide comprises a TRIM sequence.

Preferably the candidate compound is a polypeptide comprising a TRAMand/or a TRIM sequence. Such a polypeptide preferably has at least 12amino acids, more preferably at least 19, 30, 40 or 50 amino acids andpreferably less than 200 amino acids, more preferably less than 100, 90,80, 70 or 60 amino acids. The polypeptide may be shorter, for example upto 20 or up to 30 amino acids in length. Alternatively, the candidatecompound may be a non-peptide organic or inorganic molecule.

The first polypeptide and/or said second polypeptide may a viralpolypeptide, preferably a human papillomavirus (HPV) polypeptide, morepreferably an HPV E6 polypeptide, most preferably an HPV E6 polypeptidefrom HPV strain 16 and 18. The first polypeptide and/or said secondpolypeptide may also be a polypeptide found in eukaryotic cells, forexample a polypeptide selected from transcription factors and cell cycleregulatory proteins. Preferably, the first and/or second polypeptide isselected from Mdm-2, p53, TBP, E2F, YY1, CBP/p300 and TFIIB.

A TRIM or TRAM-containing polypeptide may itself be used to disrupt aninteraction between a TRIM-containing polypeptide and a TRAM-containingpolypeptide. For example, an oligopeptide consisting essentially of theTRIM sequence of E1A or p53 or an oligopeptide consisting essentially ofthe TRAM sequence of Mdm-2 may be introduced into a tumour cellover-expressing Mdm-2 to prevent Mdm-2-mediated inhibition of thep53-mediated cell cycle arrest/apoptotic pathway resulting in death ofthe tumour cell.

Accordingly, the present invention also provides the use of a compoundin a method of disrupting an interaction between a first polypeptide anda second polypeptide, wherein said compound is a polypeptide comprisinga TRAM sequence and/or a TRIM sequence, said first polypeptide comprisesa TRAM sequence and/or said second polypeptide comprises a TRIMsequence.

The present invention further provides the use of a compound in an invitro method of disrupting an interaction between a first polypeptideand a second polypeptide, wherein said compound is a polypeptidecomprising a TRAM sequence and/or a TRIM sequence, said firstpolypeptide comprises a TRAM sequence and/or said second polypeptidecomprises a TRIM sequence.

As discussed above, the disruption of TRIM-TRAM interactions will haveclinically important applications. Thus, the present invention alsoprovides the use of a compound in the manufacture of a medicament foruse in a method of disrupting an interaction between a first polypeptideand a second polypeptide, wherein said compound is a polypeptidecomprising a TRAM sequence and/or a TRIM sequence, said firstpolypeptide comprises a TRAM sequence and/or said second polypeptidecomprises a TRIM sequence.

Preferably the uses described above are where the disruption of saidinteraction inhibits virai transcription, or inhibits cell cycleprogression in mammalian cells, preferably a cancer cell.

The present invention further provides a pharmaceutical compositioncomprising a polypeptide comprising a TRAM and/or TRIM sequence.

The present invention also provides a polypeptide in substantiallyisolated form consisting essentially of a TRAM and/or TRIM sequence.

The present invention further provides a polynucleotide moleculecomprising a coding region encoding a polypeptide of the invention,preferably a polypeptide consisting essentially of a TRAM and/or TRIMsequence. The polynucleotide may also comprise additional coding regionlinked to, and in frame with, the coding region encoding a polypeptideof the invention. Polynucleotides of the invention may also beincorporated into nucleic acid vectors to produce a nucleic acid vectorof the present invention.

TRAM/TRIM-containing polypeptides of the invention may also be used toidentify novel TRAM/TRIM-containing polypeptides, for examplepolypeptides involved in cell cycle control and/or transcriptionalregulation.

Accordingly the present invention provides a method for identifying acompound which interacts with a polypeptide comprising a TRAM sequenceand/or a TRIM sequence which method comprises:

-   -   (a) incubating a candidate compound with a polypeptide        comprising a TRAM sequence and/or a TRIM sequence under suitable        conditions; and    -   (b) determining if said candidate compound interacts with said        polypeptide comprising a TRAM sequence and/or a TRIM sequence;

DETAILED DESCRIPTION OF THE INVENTION

Unless indicated otherwise, the techniques and methodologies describedare standard biochemical techniques. Examples of suitable generalmethodology textbooks include Sambrook et al., Molecular Cloning, ALaboratory Manual (1989) and Ausubel et al., Current Protocols inMolecular Biology (1995), John Wiley & Sons, Inc.

A. Polypeptides

Polypeptides of the invention consist essentially of a TRanscriptionalAdaptor Motif (TRAM) and/or a TRAM Interaction Motif (TRIM). Preferablypolypeptides of the invention are truncated variants of full-lengthTRAM- and/or TRIM-containing polypeptides, but may be such a full-lengthpolypeptide. Thus, for example, polypepuides of the invention mayconsist essentially of at least 12 amino acids, preferably at least 19amino acids, more preferably at least 30, 40 or 50 amino acids, butpreferably less than 200 amino acids, more preferably less than 150amino acids, most preferably less than 100, 90, 80, 70 or 60 aminoacids. The polypeptide may be shorter, for example up to 20 or up to 30amino acids in length.

Polypeptides of the invention may, however, be part of a largerpolypeptide, for example a fusion protein. In this case, the additionalpolypeptide sequences are preferably polypeptide sequences with whichthe polypeptide of the invention is not normally associated.

A TRAM sequence of the invention is a minimal amino acid sequence whichcan interact with a protein capable of binding to a wild-type TRAMsequence. Such wild-type sequences are the wild-type CBP, Mdm-2 and p300TRAM sequences noted below. Variants of a wild-type sequence may thus beused to provide specific instructions with a subset of the proteins thatnormally bind the wild-type TRAM.

A suitable TRAM sequence is one which binds the consensus TRIM sequenceFX[E/D]XXXL (leucine can be replaced with a similar neutral non-polarresidue such as isoleucine, valine, methionine or phenylalanine).Preferably, a TRAM sequence consists essentially of the following:

-   -   (i) two or more basic residues at, or near such as within four,        three or two amino acid residues of, the N-terminus;    -   (ii) a cysteine-proline-valine/isoleucine-cysteine (CP[V/I]C)        sequence, which is preferably part of a zinc finger motif;    -   (iii) an asparagine (N) residue between (i) and (ii); and    -   (iv) a basic residue immediately following the CP[V/I]C        sequence.

More preferably, the TRAM sequence also contains an isoleucine residueat, or near such as within four, three or two amino acid residues of,the C-terminus. Particularly preferred examples of TRAM sequences of theinvention are polypeptides consisting essentially of:

-   -   [K/R, K/R] XNXXCP [V/I] C [K/R] X (SEQ ID NO. 1)    -   [K/R, K/R] XNXXCP [V/I] C [K/R] XI (SEQ ID NO. 2)    -   RKTNGGCPVCKQ (SEQ ID NO. 3—derived from CBP)    -   RKTNGGCPVCKQPI (SEQ ID NO. 4—derived from CBP)    -   GCKRKTNGGCPVCKQLIAL (SEQ ID NO. 5—derived from CBP)    -   KKRNKPCPVCRQ (SEQ ID NO. 6—derived from Mdm-2)    -   KKRNKPCPVCRQPI (SEQ ID NO. 7—derived from Mdm-2)    -   RKTNGGCPICKQ (SEQ ID NO. 8—derived from p300)    -   RKTNGGCPICKQLI (SEQ ID NO. 9—derived from p300)

SEQ ID NOS: 3 to 9 are wild-type TRAM sequences. Examples ofTRIM-containing polypeptides which may be used to determine whether acandidate TRAM sequence functions as such include E1A, E2F, p53, TFIIB,YY1 and MyoD and certain HPV E6 variants. Full-length CBP, Mdm-2 or p300may be used as the first, TRAM-containing, polypeptide in the presentinvention.

A TRIM sequence of the invention is a minimal amino acid sequence whichcan bind the consensus TRAM sequence [K/R, K/R] XNXXCP [V/I] C [K/R] X.A TRIM sequence may thus bind the consensus TRAM sequence[K/R,K/R]XNXXCP[V/I]C[K/R]XI or one or more of the wild-type TRAMsequences. A TRIM sequence is thus capable of binding a polypeptidecontaining such a TRAM sequence.

A suitable TRIM sequence consists essentially of the consensus sequenceFX[E/D]XXXL (leucine can be replaced with a similar neutral non-polarresidue such as isoleucine, valine, methionine or phenylalanine).Particularly preferred examples of TRIM sequences of the invention arepolypeptides consisting essentially of:

-   -   FX[E/D]XXXL (SEQ ID NO. 10)    -   FPESLIL (SEQ ID NO. 11—derived from E1A)    -   FSDLWKL (SEQ ID NO. 12—derived from p53)    -   FKEITTM (SEQ ID NO. 13—derived from TFIIB)    -   FEDQILI (SEQ ID NO. 14—derived from YY1)    -   FRDNSAM (SEQ ID NO. 15—derived from YY 1)    -   FVESSKL (SEQ ID NO. 16—derived from YY 1)    -   FYDDPCF (SEQ ID NO. 17—derived from MyoD)

A TRIM sequence is also located with the second zinc finger of HPV-16 or-18 E6 protein, in particular between HPV-16 E6 residues 100 to 147 andbetween the corresponding residues of HPV-18 E6 protein. Examples ofTRAM-containing polypeptides which may be used to determine whether acandidate TRIM sequence functions as such include CBP, p300 and Mdm-2.Full length E1A, p53, TFIIB, YYI, MyoD, HPV-16 E6 or HPV-18 E6 may beused as the second TRIM-containing, polypeptide in the invention.

Polypeptides comprising TRIM/TRAM sequences may be modified to providepolypeptides of the invention. Amino acid substitutions may be made, forexample from 1, 2 or 3 to 10, 20 or 30 substitutions provided that themodified polypeptide retains substantially similar TRIM/TRAM bindingactivity (for example at least 70, 80 or 90% of the binding activity ofthe non-modified polypeptide). Amino acid substitutions may include theuse of non-naturally occurring analogues, for example to increase bloodplasma half-life of a therapeutically administered polypeptide.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

Alternatively, mutations may be made which reduce or abolish the bindingability of TRIM/TRAM sequences to produce derivatives which may be used,for example, to study the role of a TRIM/TRAM-containing polypeptide incell cycle control and/or viral infection.

In addition, polypeptides of the invention may be cyclized, for exampleas described in U.S. Pat. No. 5,723,575. Cyclization of polypeptides, inparticular small peptides, can be used to confer conformationalconstraints on such peptides, which may be advantageous in drug design.

Polypeptides of the invention may be in a substantially isolated form.It will be understood that the polypeptide may be mixed with carriers ordiluents which will not interfere with the intended purpose of thepolypeptide and still be regarded as substantially isolated. Apolypeptide of the invention may also be in a substantially purifiedform, in which case it will generally comprise the polypeptide in apreparation in which more than 90%, e.g. 95%, 98% or 99%, by weight ofthe polypeptide in the preparation is a polypeptide of the invention.

Polypeptides of the invention may be made by synthetic means orrecombinantly using techniques well known to skilled persons.Polypeptides of the invention may also be produced as fusion proteins.Examples of fusion protein partners include glutathione-S-transferase(GST), 6×His, GAL4 (DNA binding and/or transcriptional activationdomains) and β-galactosidase.

Polypeptides of the invention may be used in in vitro or in vivo cellculture systems to study the role of TRIM/TRAM interactions in cellularcontrol mechanisms, particularly with respect to the ways in which viralTRIM/TRAM containing polypeptides circumvent such control mechanisms,for example the E6 protein of HPV. For example, TRIM/TRAM polypeptidesmay be introduced into a cell to disrupt the normal functions that occurin the cell. They may also be introduced into a cell before, concomitantwith, or after viral infection to determine if virus growth andpropagation can be inhibited. The polypeptides of the invention may beintroduced into the cell by in situ expression of the polypeptide from arecombinant expression vector (see below). The expression vectoroptionally carries an inducible promoter to control the expression ofthe polypeptide.

The use of mammalian host cells is expected to provide for suchpost-translational modifications (e.g. myristolation, glycosylation,truncation, lapidation and tyrosine, serine or threoninephosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. Such cell culturesystems in which polypeptides of the invention are expressed may be usedin assay systems to identify candidate substances which interfere withor enhance the functions of TRIM/TRAM containing polypeptides in thecell.

Polypeptides of the invention may also be used to produce antibodiesagainst the TRIM or TRAM sequences. Antibodies may be polyclonal ormonoclonal. Techniques for producing antibodies are well known topersons skilled in the art (see for example, Harlow and Lane, 1988.Antibodies: .A Laboratory Manual. CSH Laboratory. Cold Spring Harbor,N.Y.)

B. Polynucleotides and Vectors

Polynucleotides of the invention comprise nucleic acid sequencesencoding the polypeptides of the invention. Polynucleotides of theinvention may comprise DNA or RNA. They may also be polynucleotideswhich include within them synthetic or modified nucleotides. A number ofdifferent types of modification to oligonucleotides are known in theart. These include methylphosphonate and phosphorothioate backbones,addition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the polynucleotides described herein may be modified byany method available in the art. Such modifications may be carried outin order to enhance the in vivo activity or life span of polynucleotidesof the invention.

Preferred polynucleotides of the invention include polynucleotidesencoding any of the polypeptides of the invention described above. Itwill be understood by a skilled person that numerous differentpolynucleotides can encode the same polypeptide as a result of thedegeneracy of the genetic code.

Polynucleotides of the invention can be incorporated into a recombinantreplicable vector. The vector may be used to replicate the nucleic acidin a compatible host cell. Thus in a further embodiment, the inventionprovides a method of making polynucleotides of the invention byintroducing a polynucleotide of the invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell. Suitable host cells includebacteria such as E. coli, yeast, mammalian cell lines and othereukaryotic cell lines, for example insect Sf9 cells.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence which is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A control sequence“operably linked” to a coding sequence is ligated in such a way thatexpression of the coding sequence is achieved under condition compatiblewith the control sequences.

Such vectors may be transformed or transfected into a suitable host cellas described above to provide for expression of a polypeptide of theinvention. This process may comprise culturing a host cell transformedwith an expression vector as described above under conditions to providefor expression by the vector of a coding sequence encoding thepolypeptides, and optionally recovering the expressed polypeptides.

The vectors may be for example, plasmid or virus vectors provided withan origin of replication, optionally a promoter for the expression ofthe said polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. Vectors may be used invitro, for example for the production of RNA or used to transfect ortransform a host cell. The vector may also be adapted to be used invivo, for example in a method of gene therapy.

Promoters/enhancers and other expression regulation signals may beselected to be compatible with the host cell for which the expressionvector is designed. For example, mammalian promoters, such as β-actinpromoters, may be used. Tissues-specific promoters are especiallypreferred. Viral promoters may also be used, for example the Moloneymurine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcomavirus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus(CMV) IE promoter, herpes simplex virus promoters or adenoviruspromoters. All these promoters are readily available in the art.

C. Viral Vectors

The polynucleotides of the invention may be used in the form of a nakednucleic acid construct. Alternatively, they may be introduced into avariety of nucleic acid vectors. Such vectors include plasmids and viralvectors, preferably herpes simplex virus vectors. Vectors may furtherinclude sequences flanking a polynucleotide of the invention whichcomprise sequences homologous to eukaryotic genomic sequences,preferably mammalian genomic sequences, or viral genomic sequences. Thiswill allow the introduction of the polynucleotides of the invention intothe genome of eukaryotic cells or viruses by homologous recombination.In particular, a plasmid vector comprising the polynucleotide of theinvention flanked by viral sequences, can be used to prepare a viralvector, suitable for delivering the polynucleotides of the invention toa mammalian cell. The techniques employed are well-known to a skilledperson. Examples of suitable viral vectors include herpes simplex virusvectors and viral vectors able to integrate their genomes into the hostcell genome, for example retroviruses, including lentiviruses, andadeno-associated virus.

D. Candidate Substances for Use in Assay Methods

Interactions between TRAM-containing polypeptides (for example Mdm-2 orCBP) and TRIM-containing polypeptides (for example p53) are important incell cycle regulation, transcriptional regulation and viral infection.The identification, reported here, of the minimal consensus motifsthrough which these interactions are mediated, has importantimplications in several areas, for example drug design. In particular,nearly all protein-protein interaction surfaces identified to date havebeen quite large. This has made it difficult to identify small organicor inorganic molecules that can disrupt such interactions. The TRIM andTRAM motifs identified here represent some of the smallest interactioninterfaces described to date. Thus a screening approach using peptidescontaining these motifs is likely to be more successful in identifyinglow molecular weight compounds capable of disrupting the protein-proteininteractions than screens based on more extensive interaction surfaces.Identification of substances which disrupt an interaction between the aTRAM-containing polypeptide and a TRIM-containing polypeptide may resultin the development of drugs which can modulate transcription, cell cyclecontrol and viral infection in a therapeutically useful way. Inaddition, the knowledge that these TRAM and TRIM motifs are important inthese interactions will allow identification of other components ofcellular and viral machinery that are involved in the processes oftranscription, cell cycle control and viral infection.

A substance which disrupts an interaction between a TRAM-containingpolypeptide (a first polypeptide) and a TRIM-containing polypeptide (asecond polypeptide) may do so in several ways. It may directly disruptthe binding of the two components by, for example, binding to onecomponent and masking or altering the site of interaction with the othercomponent. Candidate substances of this type may conveniently bescreened by in vitro binding assays as, for example, described below.Examples of candidate substances include polypeptides containing TRIMand/or TRAM sequences, other organic and inorganic low molecular weightcompounds as well as antibodies which recognize the first or secondpolypeptides.

Candidate TRIM and/or TRAM containing polypeptides may conveniently beidentified using database searches to locate polypeptide sequences whichmatch the TRIM and/or TRAM consensus sequences given in, for example SEQID. NO. 1 or 9, or which have homology to the TRIM and/or TRAM sequencesidentified in actual polypeptides, for example Mdm2, CBP, E6 and p53.

A substance which can bind directly to the first or second component mayalso inhibit an interaction between the first polypeptide and the secondpolypeptide by altering their subcellular localisation thus preventingthe two components from coming into contact within the cell. This can betested in vivo using, for example the in vivo assays described below.The term ‘in vivo’ is intended to encompass experiments with cells inculture as well as experiments with intact multicellular organisms.

Alternatively, instead of preventing the association of the componentsdirectly, the substance may suppress or enhance the biologicallyavailable amount of one or both of the components. This may be byinhibiting expression of the component, for example at the level oftranscription, transcript stability, translation or post-translationalstability. An example of such a substance would be antisense RNA whichsuppresses the amount of first or second polypeptide mRNA translatedinto protein.

Suitable candidate substances include peptides comprising TRIM and/orTRAM sequences, especially of from about 12 to 20 amino acids in size.Peptides from panels of peptides comprising random sequences orsequences which have been varied consistently to provide a maximallydiverse panel of peptides may be used. Cyclized versions of thesepeptides may also be used.

Suitable candidate substances also include antibody products (forexample, monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies and CDR-grafted antibodies) which are specific forthe first component or the second component. Furthermore, combinatoriallibraries, peptide and peptide mimetics, defined chemical entities,oligonucleotides, and natural product libraries may be screened foractivity as inhibitors of an interaction between the first polypeptideand the second polypeptide in assays such as those described below. Thecandidate substances may be used in an initial screen in batches of, forexample 10 substances per reaction, and the substances of those batcheswhich show inhibition tested individually. Candidate substances whichshow activity in in vitro screens such as those described below can thenbe tested in in vivo systems, such as mammalian cells which will beexposed to the inhibitor and tested for susceptibility to viralinfection or apoptosis as appropriate.

An important observation, detailed in Example 1, is that TRAM variantscan show differential binding to different TRIM sequences. Therefore, itwill be desirable to test variants of TRAM sequences or TRIM sequencesto determine specificity. This will be especially important fortherapeutic applications where specificity is likely to be a criticalconsideration.

E. Assays

The assay methods of the invention may be in vitro assays or in vivoassays, for example using an animal model. One type of in vitro assayfor identifying substances which disrupt an interaction between thefirst polypeptide (containing a TRAM sequence) and the secondpolypeptide (containing a TRIM sequence) involves:

-   -   contacting a first polypeptide, which is immobilised on a solid        support, with a non-immobilised second polypeptide in the        absence of a candidate substance;    -   contacting the first immobilised polypeptide with the        non-immobilised second polypeptide in the presence of a        candidate substance; and    -   determining if the candidate substance disrupts the interaction        between the first polypeptide and the second polypeptide.

Alternatively, the second polypeptide may be immobilised and firstpolypeptide non-immobilised.

In a preferred assay method, the first polypeptide is immobilised onbeads such as agarose beads. Typically this is achieved by expressingthe component as a GST-fusion protein in bacteria, yeast or highereukaryotic cell lines and purifying the GST-fusion protein from crudecell extracts using glutathione-agarose beads (Smith and Johnson, 1988).As a control, binding of the second polypeptide, which is not aGST-fusion protein, to the immobilised first polypeptide is determinedin the absence of the candidate substance. The binding of the secondcomponent to the immobilised first polypeptide is then determined in thepresence of the candidate substance. Any inhibitory effect by thecandidate substance can then be evaluated. This type of assay is knownin the art as a GST pulldown assay (see methods).

The candidate substance may be pre-incubated with the first polypeptideor with the second polypeptide or added to the reaction mixture afterpre-incubation of the first polypeptide with the second polypeptide. Ina similar assay, the second polypeptide is a GST fusion proteinimmobilised on glutathione agarose beads and the first polypeptide isnot a GST-fusion protein. It is also possible to perform this type ofassay using different affinity purification systems for immobilising oneof the polypeptides, for example Ni-NTA agarose and histidine-taggedpolypeptides, MBP-tagged polypeptides. Alternatively, polypeptides maybe immobilised by covalent linkage via, for example, activated cyanogenbromide.

Binding of the first polypeptide to the second polypeptide (andvice-versa) may be determined by a variety of methods well-known in theart. For example, the non-immobilised polypeptide may be labelled (withfor example, a radioactive label, an epitope tag or an enzyme-antibodyconjugate). The effect of a candidate substance on an interactionbetween the two polypeptides can be determined by comparing the amountof label bound in the presence of the candidate substance with theamount of label bound in the absence of candidate substance. A loweramount of label bound in the presence of the candidate substanceindicates that the candidate substance is an inhibitor of interactionsbetween the first polypeptide and the second polypeptide. For example,typically for a candidate substance to be selected as an inhibitor ofthe interaction between the first and the second polypeptide, the amountof second polypeptide which binds to the first polypeptide (asindicated, for example, by the amount of bound label) in the presence ofthe inhibitor is at least 50%, preferably at least 75%, more preferablyat least 80, 90, 95%, of the amount of second polypeptide which binds tothe first polypeptide in the absence of the inhibitor. Similarconsiderations will apply to the various assays systems used, forexample where binding is determined by transcriptional activation (asdescribed below) or in a functional in vivo assay such as an assay whichmeasures the degree of p53-mediated apoptosis or cell cycle arrest.

Alternatively, binding may be determined by immunological detectiontechniques. For example, the reaction mixture can be Western blotted andthe blot probed with an antibody that detects the non-immobilisedpolypeptide. ELISA techniques may also be used.

Another method contemplated by the invention for identifying a substancethat disrupts an interaction between the first polypeptide and thesecond polypeptide involves immobilising the first polypeptide on asolid support coated (or impregnated with) a fluorescent agent,labelling the second polypeptide with a substance capable of excitingthe fluorescent agent, contacting the immobilised first polypeptide withthe labelled second polypeptide in the presence and absence of a testcompound, detecting light emission by the fluorescent agent, andidentifying inhibitory substances as those candidate substances thatreduce the emission of light by the fluorescent agent in comparison tothe emission of light by the fluorescent agent in the absence of thetest compound. Alternatively, the second polypeptide may be immobilisedand the first polypeptide labelled in the assay.

Assays for identifying compounds that disrupt an interaction between thefirst and second polypeptide may also involve:

-   -   (a) transforming or transfecting an appropriate host cell with a        DNA construct comprising a reporter gene under the control of a        promoter regulated by a transcription factor having a        DNA-binding domain and an activating domain;    -   (b) expressing in the host cell a first hybrid DNA sequence        encoding a first fusion of the first polypeptide and the DNA        binding domain or the activating domain of the transcription        factor; expressing in the host cells a second hybrid DNA        sequence encoding the second polypeptide and the DNA binding        domain or activating domain of the transcription factor which is        not incorporated in the first fusion;    -   (c) evaluating the effect of a test compound on the interaction        between the first polypeptide and the second polypeptide by        detecting binding of the first polypeptide to the second        polypeptide in a particular host cell by measuring the        production of reporter gene product in the host cell in the        presence or absence of the test compound; and    -   (d) determining whether the presence of the test compound alters        the production of the reporter gene product in comparison to the        production of the reporter gene product in the absence of the        test compound.

The host cell may be a bacterium or other microbial cell. It may be ayeast or mammalian cell. Presently preferred for use in such an assayare a lexA promoter to drive expression of the reporter gene, the lacZreporter gene, a transcription factor comprising the lexA DNA domain andthe GAL4 transactivation domain and yeast host cells.

The candidate substance, i.e. the test compound, may be administered tothe cell in several ways. For example, it may be added directly to thecell culture medium or injected into the cell. Alternatively, in thecase of polypeptide candidate substances, the cell may be transfectedwith a nucleic acid construct which directs expression of thepolypeptide in the cell. Preferably, the expression of the polypeptideis under the control of a regulatable promoter.

TRIM and/or TRAM containing polypeptides and candidate substances thatare identified by the method of the invention as disrupting aninteraction between a first polypeptide and a second polypeptide may betested for their ability to, for example, reduce susceptibility of cellsto viral infection or regulate the cell cycle including apoptosis andgrowth arrest. Such compounds could be used therapeutically to preventor treat viral infection. They may also be used therapeutically inregulating the cell cycle of a mammalian cell, including inducing celldeath in, for example, tumour cells.

Typically, an assay to determine the effect of a candidate substance(which may be a TRIM and/or TRAM-containing polypeptide or a substanceidentified by the method of the invention) on the susceptibility ofcells to viral infection comprises:

-   -   (a) administering a virus, for example HPV, to a cell, for        example primary human keratinocytes, in the absence of the        candidate substance;    -   (b) administering the virus to the cell in the presence of the        candidate substance; and    -   (c) determining if the candidate substance reduces or abolishes        the susceptibility of the cell to viral infection.

The candidate substance may be administered before, or concomitant with,the virus to establish if infection is prevented. Alternatively, thecandidate substance may be administered subsequent to viral infection toestablish if viral infection can be treated using the candidatesubstance. Administration of candidate substances to cells may beperformed as described above.

The assay is typically carried out in vitro but an animal model could beemployed instead. The virus is contacted with cells, typically cells inculture. The cells may be cells of a mammalian cell line, in particularmammalian cells susceptible to infection by the virus in the absence ofthe candidate substance.

Techniques for assaying infectivity of viruses are well-known in theart. As well as using plaque assays, levels of viral infection can bedetermined by using recombinant viruses which comprise a reporter gene,for example lacZ. The use of a histochemically detectable reporter geneis especially preferred when experiments are performed with animals, forexample mice.

Typically, an assay to determine the effect of a TRIM and/orTRAM-containing polypeptide or candidate substance identified by themethod of the invention on the regulation of the cell cycle in amammalian cell comprises:

-   -   (a) administering the candidate substance to the cell; and    -   (b) determining the effect of the candidate substance on the        cell cycle, including, for example induction of cell cycle        arrest and/or cell death by apoptosis.

Administration of candidate substances to cells may be performed asdescribed above. The assay is typically carried out in vitro. Thecandidate substance is contacted with the cells, typically cells inculture. The cells may be cells of a mammalian cell line.

The ability of a candidate substance to induce apoptosis can bedetermined by administering a candidate compound to cells anddetermining if apoptosis is induced in said cells. The induction ofapoptosis can be determined by various means. There are severaltechniques known to a skilled person for determining if cell death isdue to apoptosis. Apoptotic cell death is characterized by morphologicalchanges which can be observed by microscopy, for example cytoplasmicblebbing, cell shrinkage, internucleosomal fragmentation and chromatincondensation. DNA cleavage typical of the apoptotic process can bedemonstrated using TUNEL and DNA ladder assays.

Several techniques known in the art for inducing apoptosis in cells maybe used. For example, apoptosis may be induced by stress including UV orgamma irradiation exposure, growth factor deprivation and heat shock.The ability of the candidate substance to inhibit such apoptosis may bedetermined by comparing cells exposed to stress in the presence of thecandidate substance with those exposed to stress in the absence of thecandidate substance.

In a preferred embodiment of the above-described assays, TRIM and/orTRAM containing polypeptides, or derivatives thereof are used in anexperimental system to study normal cellular interactions. For example,polypeptides containing derivatives of TRIM and/or TRAM sequences,including deletion, insertion and substitution mutants, can be used todisrupt an interaction between a TRIM-containing polypeptide and/or aTRAM-containing polypeptide. This can be tested in vitro using the invitro assays described above. These interaction can also be disrupted invivo by introducing TRIM and/or TRAM containing polypeptides andderivatives thereof, including deletion, insertion and substitutionmutants, into cells in vivo, preferably mammalian cells, more preferablyhuman cells. TRIM and/or TRAM containing polypeptides and theirderivatives can be introduced into the cells using techniques describedabove. The effect of this disruption can be determined usingimmunoprecipitation studies or, alternatively, by analyzing the effecton cell cycle control using, for example, the assays and techniquesdescribed above. Any in vitro data obtained may be used to assist in therational design of TRIM and/or TRAM containing polypeptides orderivatives thereof for use in the in vivo studies.

In addition to identifying substances which disrupt an interactionbetween a TRAM-containing polypeptide and a TRIM-containing polypeptide,the polypeptides of the invention may be used in an assay/screeningmethod to identify substances which bind to a TRAM sequence or a TRIMsequence. This may result in the identification of novel components ofcellular or viral machinery involved in cell cycle control,transcription and/or viral infection. Thus the present inventionprovides a method for identifying a compound which interacts with apolypeptide comprising a TRAM sequence and/or a TRIM sequence whichmethod comprises:

-   -   (a) incubating a candidate compound with a polypeptide        comprising a TRAM sequence and/or a TRIM sequence under suitable        conditions; and    -   (b) determining if said candidate compound interacts with said        polypeptide comprising a TRAM sequence and/or a TRIM sequence.

Typically, such assays are carried out in vitro using similar formats tothose described above (for example incubating a candidate substance witha TRIM/TRAM polypeptide immobilised on a solid phase (for example a GSTpulldown assay) and interactions determined by similar techniques to,those described above. These assays may thus be used to screen, forexample, crude or partially-purified cellular extracts for novelTRIM/TRAM polypeptides which may be part of the cellular signaltransduction, cell cycle control, transcriptional control machinery, orinvolved in viral infection. It may also be used to identify candidatesubstances for use in the assays described above.

F. Diagnostic Uses

It has been shown in the Examples that different types of humanpapillomavirus have E6 polypeptides with different TRAM-bindingproperties. For example, the E6 polypeptides of two types which areknown to be associated with a high risk of cervical cancer (HPV-16 andHPV-18) can bind the CBP TRAM sequence. They contain a consensus TRIMsequence. The E6 polypeptides of two types which are known to beassociated with a low risk of cervical cancer (HPV-6 and HPV-11) do notbind CBP TRAM sequence. They do not contain a consensus TRIM sequence.Consequently, assays may be used to distinguish HPV types associatedwith a high risk of cervical cancer from HPV types associated with a lowrisk of cervical cancer on the basis of selective binding to a TRAMsequence, in particular the CBP TRAM sequence. Such assays may take theform of immunoassays or polypeptide/polypeptide binding assays.

G. Therapeutic Uses

All of the specific TRIM/TRAM polypeptides described above are part ofpolypeptides known to be involved in transcriptional regulation of genesinvolved in signal transduction and/or cell cycle control (for example,p53, Mdm-2, CBP/p300 and HPV E6). Consequently, a substance whichdisrupts an interaction between these TRIM/TRAM containing polypeptides(which may, for example, be identified by the assay methods of theinvention is likely to have an effect on transcriptional regulationand/or cell cycle control. Thus such a substance may be used to regulatetranscription and/or the cell cycle of a mammalian cell. Accordingly,the present invention provides a substance capable of disrupting aninteraction between (i) a TRAM-containing polypeptide and (ii) aTRIM-containing polypeptide for use in a method of regulating themammalian cell cycle. It also provides such a substance for use in amethod of regulating cellular transcription. Typically, said substancemay be used to induce cell death, for example in a tumour cell, or toprevent cell death. Examples of such substances include TRIM and/or TRAMcontaining polypeptides. As an example, a TRIM-containing polypeptide ora TRAM-containing polypeptide introduced into a tumour cell which isover-expressing Mdm-2 may be used to inhibit the interaction betweenMdm-2 and p53 and allow p53-mediated apoptosis to proceed.

If either or both of the TRIM/TRAM polypeptides are of viral origin,then it may be possible to inhibit or reduce interactions that arerequired for viral infection, growth and/or propagation. For example, aTRAM-containing polypeptide may be used to inhibit the binding of HPV E6to CBP. Accordingly, the present invention provides a substance capableof disrupting an interaction between (i) a TRAM-containing polypeptideand (ii) a TRIM-containing polypeptide for use in a method of preventingor treating viral infection, in particular HPV infection. In the case ofa TRAM-containing polypeptide used to inhibit the binding of HPV E6 toCBP, it is preferred to use a TRAM sequence which is specific for theTRIM sequence of E6 from HPV-16 or HPV-18 (or another HPV type known tobe associated with cervical cancer). This is because, as discussedabove, it has been shown in the Examples that the E6 polypeptides of HPVtypes which are known to be associated with a high risk of cervicalcancer (HPV-16 and HPV-18) can bind a CBP TRAM sequence whereas the E6polypeptides of two HPV types which are known to be associated with alow risk of cervical cancer (HPV-6 and HPV-11) do not bind the CBP TRAMsequence.

The formulation of a substance according to the invention will dependupon the nature of the substance, for example whether it is apolypeptide or a non-peptide organic or inorganic molecule, buttypically a substance may be formulated for clinical use with apharmaceutically acceptable carrier or diluent. For example it mayformulated for topical, parenteral, intravenous, intramuscular,subcutaneous, intraocular or transdermal administration. A physicianwill be able to determine the required route of administration for anyparticular patient and condition.

The polynucleotides of the invention may be administered directly as anaked nucleic acid construct, preferably further comprising flankingsequences homologous to the host cell genome. Uptake of naked nucleicacid constructs by mammalian cells is enhanced by several knowntransfection techniques for example those including the use oftransfection agents. Example of these agents include cationic agents(for example calcium phosphate and DEAE-dextran) and lipofectants (forexample lipofectam™ and transfectam™). Typically, nucleic acidconstructs are mixed with the transfection agent to produce acomposition.

Preferably, the substance is used in an injectable form. It maytherefore be mixed with any vehicle which is pharmaceutically acceptablefor an injectable formulation, preferably for a direct injection at thesite to be treated. The pharmaceutically carrier or diluent may be, forexample, sterile or isotonic solutions. It is also preferred toformulate that substance in an orally active form. Typically, saidsubstance may be a polypeptide, an antibody or a nucleic acid construct.Nucleic acid constructs may be administered by various well-knowntechniques including lipofection, biolistic transformation or the use ofviral vectors. Virus-like particles (VLPs) may also be used to deliverpolynucleotides of the invention. VLPs can be generated by techniquesknown in the art. For example, VLPs formed in cell culture from theover-expression of the L1 and L2 genes of human papillomavirus type-33in Cos-7 cells have been shown to efficiently incorporatepolynucleotides (Unckell et al., 1997). The use of papillomavirus VLPsfor delivering TRIM and/or TRAM containing polypeptides to cells wouldbe particularly desirable for disrupting high risk E6 polypeptideinteractions with CBP, Mdm2 or other TRAM-containing polypeptides sincethe VLPs would demonstrate a tropism similar to that of the naturalpapillomavirus infection.

The dose of substance used may be adjusted according to variousparameters, especially according to the substance used, the age, weightand condition of the patient to be treated, the mode of administrationused and the required clinical regimen. A physician will be able todetermine the required route of administration and dosage for anyparticular patient and condition.

The invention will be described with reference to the following Exampleswhich are intended to be illustrative only and not limiting. TheExamples refer to the Figures. Referring to the Figures in more details:

FIGS. 1 a-1 d. E1A binds a 12 amino acid motif in CBP (amino acids1811-1822).

FIG. 1 a, schematic representation of the transcription factor CBPshowing an amino acid region (between 1621-1877) that binds the factorsE1A, p53, E2F, and TFIIB. FIG. 1 b, GST-CBP fusion constructs used inpull-down experiments to define sequences capable of binding 12S E1A.The bacterial GST-fusion proteins were bound to micro-columns and invitro translated ³⁵S-labeled E1A passed over them to detectprotein-protein interactions (see methods). Approximately 10% of the E1Atranslation reaction was run in the input lane, GST represents a controlcolumn, and lanes 1 to 9 represent the eluate obtained after passing E1Aover columns containing the nine GST-CBP fusion constructs. FIG. 1 c,Further deletion analysis of CBP sequences 1808-1826. Thick linesindicate constructs that bind E1A, narrow lines those that do not. Aminimal construct of 12 amino acids (construct 10, CBP 1811-22) stillretains E1A binding activity. FIG. 1 d, Mutagenesis of the CBP TRAM.Indicated are the eleven alanine substitutions used to determine aminoacids required for the E1A interaction. All are single substitutions,except for mutant construct 1, which substitutes two basic residues.Pull-down experiments using wild-type or mutant GST-CBP (1808-1826)sequences were carried out and show that E1A binding is abolished by theR1811A, K1812A mutation and the N1814A mutation. Also shown is theamount of GST-CBP protein used in the pull-down experiment.

FIGS. 2 a-2 d. Identification of the amino acids in E1A involved in theinteraction with CBP.

FIG. 2 a, schematic representation of 13S E1A depicting conservedregions CR1, CR2, and CR3. Contained within CR1 are residues 63-67,previously implicated in the binding of CBP. GST-E1A proteins containingwild type or mutated (alanine substituted) sequences were tested fortheir ability to interact with ³⁵S-labeled CBP (1621-1877). Shown arethe results of these pull-down experiments along with a picture of acoomassie-stained SDS PAGE gel to show the quantities of GST-E1A fusionproteins recovered from the micro-columns. Double substitution mutantswithin sequences F65 to L71 fail to bind CBP. FIG. 2 b, Peptidecompetition assays confirm the involvement of sequences F65 to L71 inthe interaction with CBP. Peptides of 30 amino acids containing thesequences shown above were used in competition studies to test theirability to prevent the E1A-CBP interaction. Increasing amounts (0.1 mM,0.5 mM, and 1 mM) of wild type peptide sequence (WT), or mutantsequences (Mut 1 or Mut 2) were used in E1A-CBP pull-down assays. Onlythe wild type peptide prevented the E1A-CBP interaction. FIG. 2 c, p53,E2F, and TFIIB all bind to CBP sequences 1808-1826 that contain theTRAM. Binding can be inhibited by competition with a wild-type E1Apeptide, but not by the Mut 2 E1A peptide. FIG. 2 d, An alignment ofE1A, p53, and E2F sequences with the conserved FXE/DXXXL TRAMinteraction motif (TRIM) underlined.

FIGS. 3 a-3 d. Mdm2 contains a C-terminal TRAM that binds E1A, p53, E2F,and TFIIB.

FIG. 3 a, An alignment of sequences from human Mdm2 (466-484) and CBP(1808-1826). Conserved sequences are boxed and residues previouslymutated (FIG. 1 d) to prevent E1A interaction are denoted by anasterisk. FIG. 3 b, In vitro binding of E1A, p53, E2F, and TFIIB to theMdm2 TRAM. Pull-down experiments show all four in vitro translated andradiolabeled proteins bind the wild type Mdm2 TRAM contained withinsequences 466-484. A mutant GST-Mdm2 construct (N472A) fails to bindE1A, like its CBP counterpart (FIG. 1 d). FIG. 3 c, The C-terminal Mdm2TRAM is masked by N-terminal sequences between 391-421. Shown is aschematic representation of full-length Mdm2 containing the N- andC-terminal regions capable of binding p53, along with constructs used inin vitro pull-down experiments. GST-Mdm2 fusion constructs containingMdm2 sequences 223-491 and 391-491 show vastly reduced bindingcapabilities compared to the unmasked C-terminal region 421-491. Alsoshown for comparison is the use of the N-terminal 1-125 amino acidregion of Mdm2, previously shown to interact with both p53 and E2F. FIG.3 d, In vivo interaction between Mdm2 C-terminal sequences (421-491)containing an unmasked TRAM, and p53. MRC5.SV40 cells were transfectedwith 0.1 μg or 0.5 μg of either a pCMV-GST-Mdm2 (421-491) construct, ora control CMV-GST construct. After 48 hours, cell lysates were preparedfrom these cells and incubated with glutathione-Sepharose beads, thensubjected to washes and elution as described for in vivo pull-downs (seemethods). Eluted proteins were run on SDS polyacrylamide gels andsubjected to standard western blot analysis using the p53 monoclonalantibody DO-1. (Santa Cruz). Lysate containing transfected GST-Mdm2(421-491) indicated complex formation between p53 and Mdm2 (421-491),while those from the GST control transfections failed to pull-down p53protein. Lysate approximating to 10% of the amount loaded ontomicro-columns was loaded directly onto the same SDS polyacrylamide geland subjected to the same western blot analysis. A comparison betweenp53 levels detected suggests that approximately 2-3% of total cellularp53 is complexed with the GST-Mdm2 fusion protein.

FIGS. 4 a-4 d. The CBP and Mdm2 TRAMs compete for p53 binding with theN-terminal domain of full-length Mdm2 and activate p53-dependenttranscription.

FIG. 4 a, Differential effect of the p53 mutant 14/19 on the Mdm2N-terminal domain and the CBP and Mdm2 TRAMs. In vitro translated³⁵S-methionine labelled p53, either wild type or harbouring theL14Q/F19S mutation, were analysed for their ability to bind toGST-fusion proteins containing the N-terminal Mdm2 domain (1-125), theCBP TRAM (1715-1852), or the Mdm2 TRAM (421-491) in pull-down assays.While the binding of p53 to the N-terminal domain of Mdm2 is drasticallyaffected by the 14/19 mutation, neither the CBP nor the Mdm2 TRAMs areaffected. FIG. 4 b, A CBP TRAM peptide can inhibit the binding of theN-terminal Mdm2 domain to p53. CBP peptides of 27 amino acids(1806-1832) containing either wild type or mutant (R1811, K1812, N1814)TRAM sequences were used in competition assays to prevent theinteraction of in vitro translated p53 and GST-Mdm2 (1-125). Wild typepeptide was able to completely inhibit the p53-Mdm2 interaction over therange used (10-100 μM), while the ability of mutant TRAM peptide toinhibit the interaction was severely impaired. FIG. 4 c, CBP sequencescontaining a wild type TRAM activate p53-dependent transcription.Transient transfection of U-2 OS cells was carried out using either 2 μgof a p53-responsive reporter gene (PG13CAT) or a control vector(MG15CAT). Also indicated is the co-transfection of 1, 2 or 4 μg of aCMV-GST-CBP (1808-1852) vector containing either a wild type TRAM, or amutant TRAM (R1811A, K1812A). Introduction of the wild type TRAMresulted in a dose-dependent increase in p53-dependent transcription.This level of activation was not obtained when the mutant TRAM constructwas used. The co-transfection of full-length Mdm2 abolished TRAMactivation of p53 by CBP. FIG. 4 d, U-2 OS transfection experimentsdemonstrate that introduction of unmasked Mdm2 TRAM sequences result ina similar activation of p53-dependent transcription. In addition toPG13CAT, co-transfections were carried out using 2 μg of theCMV-GST-Mdm2 expression vector (containing Mdm2 sequences 421-491,391-491, 223-491, 1-491, or 1-125). It can be seen that transcriptionfrom the p53-dependent reporter construct is activated uponco-transfection of the Mdm2 421-491 construct that contains a functionalTRAM. This effect is reduced upon the inclusion of N-terminal maskingsequences (see 391-491 and 223-491). Co-transfection of the N-terminalmotif of Mdm2 alone also activated p53-dependent transcription, whilefull-length Mdm2, containing sequences that lead to p53 degradation,resulted in a reduction in p53-dependent reporter activity.

FIGS. 5 a-5 c. YY1 fragments containing sequences showing markedsimilarity to previously defined TRIMs are able to interact with CBP invitro and are capable of repressing AP-1 activity in vivo.

FIG. 5 a, Schematic representation of the YY1 constructs used in invitro pull-down assays and in in vivo repression assays. WTYY1represents the full-length wild type YY1 sequence, YY1BP, YY1BS, andYY1BH, start at amino acid position 1 and terminate at uniquerestriction sites within the YY1 sequence, while YY1ZF 2-4 and 1-4represent PCR amplified YY1 sequences containing either all 4 zincfingers (ZF 1-4) or the last 3 zinc fingers (2-4). These sequences wereeither fused to GST or GAL4 (1-147) sequences for the assays describedbelow. FIG. 5 b, In vitro pull-down assays demonstrate the ability offragments containing at least one putative TRIM sequence to interactwith IVT CBP (1621-1877), while the fragment YY1BP which does notcontain a TRIM sequence, fails to interact with CBP. FIG. 5 c, YY1fragments capable of binding CBP in vitro demonstrate an ability torepress AP-1 activity in vivo. The schematic drawing shows the twopalindromic GAL4 binding sites that were used to replace naturallyoccuring YY1 sites in the p80:2e/9e CAT reporter construct (O'Connor elal., 1996). This p80:2e/9eGAL reporter construct was then co-transfectedwith plasmids capable of expressing GAL-YY1 fusion proteins under thedirection of an SV40 promoter in primary human keratinocytes. It can beseen that the expression of GAL-YY1 fusion constructs containing TRIMsresults in a repression of CAT activity compared to those resultsobtained when only GAL sequences are co-transfected. By contrast theco-tranfection of a GAL-YY1BP construct which possess no TRIM fails toshow any significant repression of CAT activity

FIGS. 6 a-6 b. Differential binding properties of CBP TRAM mutantsequences to TRIM containing proteins.

FIG. 6 a, A sequence alignment of previously defined TRIMs along withthe putative TRIM sequences present in TFIIB, YY1 and MyoD. Thephenylalanine residue and acidic residue in postions 1 and 3respectively are highly conserved, while the leucine residue in the7^(th) position sometimes shows a conservative change with anotherneutral, non-polar amino acid residue. Also illuminated is the largevariation in amino acid residue composition in the remaining TRIMsequences, and in the surrounding sequences. FIG. 6 b, In vitropull-down assays demonstrate the failure of TRAM C-terminal residuealanine substitutions to bind a subset of TRIM-containing proteins.

FIGS. 7 a-7 d. HPV-16 E6 interacts with the transcriptional co-activatorCBP/p300. (FIG. 7 a) Equal amounts of partially purified full-length(FL) CBP/p300 from HeLa nuclear extract was passed over GST, GST-16E6,GST-P/CAF, and GST-YY1 micro-affinity columns (see Materials andMethods). After SDS-gel electrophoresis and transfer to membranes,western analysis detected the presence of CBP/p300. (FIG. 7 b) GSTmicro-affinity columns were used to detect the interaction of in vitrotranslated and radiolabeled HPV-16 E6 with GST-CBP II (residues 1621 to1877). No interaction was detected for the control GST-column or theGST-CBP I (residues 461 to 662) column. (FIG. 7 c) Comparison of theHPV-16 E6-CBP II interaction with known E1A-CBP II and HPV-16 E6-E6APinteractions using GST micro-affinity column assays. (FIG. 7 d)Demonstration of a direct interaction between HPV-16 E6 and CBP usingtwo recombinant bacterially expressed proteins. GST or GST-E6 was passedover a column containing MBP-CBP (residues 1808-1852) fusion protein.Bound GST-fusion protein was detected by western analysis using aspecific GST antibody. The MBP-CBP fusion protein was also passed over aGST or GST-E6 column and the interaction detected using an MBP antibody.

FIG. 8. Identification of an HPV-16 E6 binding site on CBP/p300. Shownis a schematic representation of GST-CBP fusion constructs used inmicro-affinity column experiments used to define CBP sequences capableof binding HPV-16 E6. Demonstrated is the ability of a 19 amino acidsequence of CBP (residues 1808-1826) to bind in vitro translated HPV-16E6 protein (lane 7). Deletion into these sequences abolishes E6 binding(lane 8). Also shown is an alignment of the identified E6-binding sitewithin CBP and a comparison with the corresponding p300 sequence. Anasterix represents the conservation of an identical amino acid residuein that position, while a “+” represents a conservative change. An E1Apeptide that can bind this 19 amino acid CBP sequence can inhibit theE6-CBP interaction, while a CBP-binding deficient mutant peptide cannot.

FIGS. 9 a-9 c. Mapping of an HPV-16 E6 region involved in theinteraction with CBP. (FIG. 9 a) Amino acid sequence of the HPV-16 E6protein indicating the two zinc finger structures present in thisprotein. Indicated are the numbers of the amino acid residues which markstart or end points of HPV-16 E6 fragments used in interaction studies.(FIG. 9 b) Schematic representation of GST-E6 fusion constructs used inmicro-affinity column assays. (FIG. 9 c) Interaction experiments definea region between HPV-16 E6 residues 100-147 as sufficient for thebinding of CBP.

FIGS. 10 a and 10 b. The E6-CBP/p300 interaction is specific for“high-risk” HPV E6 proteins. (FIG. 10 a) Micro-affinity columnexperiments using either GST-fusion or in vitro translated E6 proteinsdemonstrate that only E6 proteins from the high-risk HPV types 16 and18, but not the low-risk HPV types 6 and 11, are capable of interactingwith CBP. (FIG. 10 b) Mammalian two-hybrid experiments (described inMaterial and Methods) and shown schematically here, indicate that thedistinction between high-risk and low-risk E6 proteins extends to the invivo interaction with the CBP II domain. Activation of the G5E1BCATreporter is only seen after co-transfection of GAL4-16 E6 and CBPII-VP16, and not for those experiments in which GAL4-11 E6 or CBPI-VP16proteins were expressed.

FIGS. 11 a-11 c. The HPV-16 E6 mutant L50G binds CBP, but is unable tointeract with E6AP or degrade p53 in vitro. (FIG. 11 a) Schematicrepresentation of the HPV-16 E6 mutant L50G showing the position of theamino acid exchange in the first zinc finger (marked by a +) and theidentified CBP-interaction domain within the second zinc finger (boldline). GST micro-affinity column experiments using in vitro translatedHPV-16 E6 L50G protein demonstrate the ability of this mutant tointeract with GST-CBP. (FIG. 11 b) Similar in vitro micro-affinitycolumn experiments show that unlike the WT 16 E6 protein, but similar toHPV-11 E6, the HPV-16 E6 mutant L50G is unable to interact withGST-E6AP. (FIG. 11 c) p53 degradation assays using in vitro translated³⁵S-labeled p53 mixed with various in vitro translated E6 proteins. Thenumbered columns indicate the levels of p53 protein after variousincubation times (0, 30, 90, and 180 min) at room temperature.

FIGS. 12 a and 12 b. HPV-16 E6 targets the ability of CBP to activatep53-dependent transcription. (FIG. 12 a) U2-OS cells were transfectedwith the p53-responsive CAT reporter (PG13) or a control vector withmutated p53 binding sites (MG15). Co-transfection of expression-vectorsfor viral proteins show that HPV proteins able to interact with CBP candown-regulate p53 transactivation to a level comparable with Ad E1A.(FIG. 12 b) Over-expression of full-length CBP in experiments similar tothose described above show that HPV proteins able to interact with CBP,including the HPV-16 L50G mutant, abolish the CBP-dependentsuperactivation of p53-dependent transcription seen with full-length CBPalone.

FIG. 13. Polylinker of pMALP.

EXAMPLE 1 Materials and Methods

Plasmids and fusion proteins. GST-CBP, GST-Mdm2, or GST-E1A constructswere obtained by cloning PCR amplified fragments or double strandedoligonucleotides into pGEX2TK (Pharmacia). The GST-Mdm2 (1-125)construct was a gift from Benjamin Li. GST-fusion proteins wereexpressed in E. coli, extracted with lysis buffer (50 mM Tris-HCl pH8.0, 0.5 mM EDTA, 5 mM DTT, 15% glycerol, 1 mg/ml lysozyme and 1 mMPMSF), and after sonication and centrifugation, stored at −70° C.CMV-GST construct pXJGST was a gift from Edward Manser and wasconstructed by inserting GST sequences into the EcoRI/BamHI sites ofpXJ40 (Xiao et al., 1991). CBP and Mdm2 sequences were then cloned intothis vector via the BamHI HindIII sites.

In vitro pull-down assays using glutathione-Sepharose micro-columns.Bacterial lysate containing GST-fusion protein was incubated withglutathione-Sepharose beads (Pharmacia) for 30 min at 4° C. in 1×NENTbuffer (100 mM NaCl, 1 mM EDTA, 0.5% NP-40, Tris-HCl, pH 8.0). Afterspinning down and washing with 1 ml 1×NENT, the beads were loaded into ayellow Gilson pipette tip containing a glass bead (BDH, cat. no.332134Y) to create a 25 μl GST micro-column. In vitro transcription andtranslation of proteins incorporating ³⁵S-methionine was performed usingTNT kits (Promega) according to the manufacturer's recommendations. 40μl of a 50 μl IVT reaction was diluted with 360 μl of IPD buffer (50 mMKCl, 40 mM Hepes, pH 7.5, 5 mM 2-β mercaptoethanol, 0.1% Tween-20, 0.5%milk) before being passed down the GST micro-column. After washing thecolumn twice with 1 ml wash buffer (IPD buffer containing 150 mM KCl),proteins were eluted from the column by adding 25 μl of 2×SDS loadingdye, heating to 95° C. for 5 min, chasing with 25 μl water, and spinningin a micro-centrifuge. Approximately half of the sample was then loadedonto a SDS polyacrylamide gel and, after running, staining, andde-staining, the gels were treated with Enlightning (NEN ResearchProducts) for 30 min before drying and exposure to autoradiographicfilm.

Peptide competition assays. To study the influence of specific peptideson protein-protein interactions in pull down experiments, GST-fusionproteins were bound to glutathione-Sepharose as described previously. Inthe case of E1A peptide competition, the washed beads were incubated in200 μl IPD buffer containing peptide (final concentration 10 μM-100 mM),and rotated at 4° C. for 1 hour. 40 μl of an in vitro translationreaction was then diluted with 60 μl IPD buffer and added to the samplebefore incubating for a further 30 min at 4° C. For the CBP TRAM peptidecompetition studies, the peptide was pre-incubated with diluted in vitrotranslation reaction for 15 min at 4° C. before incubation with theGST-fusion protein. After spinning down and washing theglutathione-Sepharose beads, proteins were eluted in 50 μl 1×SDS samplebuffer by heating the beads to 95° C. for 5 min. Approximately half ofthe sample was then run on SDS polyacrylamide gels and treated asdescribed above.

Detection of in vivo interactions between GST-Mdm2 proteins andendogenous p53. MRC5.SV40 cells were transiently transfected with 0.1μg-2.0 μg of CMV-GST constructs using lipofectin reagent (Gibco-BRL).After 48 hours, cells were harvested and resuspended in MCL Buffer (50mM Tris.Cl pH 7.6, 1 mM EDTA, 1 mM DTT, 50 mM NaF, 0.3M NaCl,0.1×Protease inhibitor cocktail (Sigma), 1.5 mM PMSF). Cells were thenlysed by sonication and cell debris pelleted by ultracentrifugation. Theextent of GST and GST-Mdm2 protein expression was determined for eachlysate by western blot analysis using anti-GST antibody B-14 (SantaCruz). Lysate containing approximately equal amounts of GST or GST-Mdm2protein (usually around 500 μg) was then mixed withglutathione-Sepharose beads in 400 μl of IPD buffer and incubated for 1hour at 4° C. GST-fusion proteins, along with interacting proteins, werepurified by spinning down the glutathione-Sepharose beads and washingtwice with 1 ml of wash buffer. Remaining proteins were eluted in 50 μl1×SDS sample buffer by heating the beads to 95° C. for 5 min. Thepresence of p53 was detected by standard western blot analysis using theanti-p53 monoclonal antibody DO-1 (Santa Cruz).

Transfections and CAT assays. U-2 OS cells or MRC5.SV40 cells (a giftfrom Dr. Peter Karran) were plated onto 10 cm-diameter culture dishesand transfected at 50-70% confluency using lipofectin reagent(GIBCO-BRL) as described previously (O'Connor et al., 1996).Chloramphenicol acetyl transferase (CAT) assays have also been describedpreviously (O'Connor et al., 1996) and the data presented representsbetween three and ten independent transfection experiments.

Results

Identification and Characterisation of a 12 Amino Acid Motif in CBP(TRAM) that Binds E1A

Previously published work has shown that a 257 cystein-rich amino acidregion of CBP spanning amino acids 1621-1877 could bind E1A, TFIIB,P/CAF, c-fos and MyoD, as well as a number of other transcriptionfactors. Using glutathione-sepharose micro-columns and a series ofGST-CBP fusion proteins we initially identified a 19 amino acid regionof CBP (1808-1826) that was sufficient for the binding of E1A (FIG. 1b). Deletion into this region abolished E1A binding (FIG. 1 b, lane 9).

Further fine deletion analysis of CBP (1808-1826) identified a 12residue sequence (1811-1822) which is sufficient for E1A binding (FIG. 1d), although this sequence binds with slightly reduced affinity comparedwith the larger, 19 residue (1808-1826) sequence. We have termed thesequence between 1811-1822 a TRanscriptional Adaptor Motif (TRAM).

To establish the residues important for TRAM function we carried out amutagenesis analysis. All except one of the residues within the CBP TRAMwere mutated to alanine. FIG. 1 d shows that only two mutationsdrastically affect the binding to E1A: a pair of basic residues (RK,1811/1812) and an asparagine residue (N 1814) at the N-terminus of themotif. The fact that no other residue had a dramatic effect on E1Abinding suggests that the C-terminus of this motif provides a lowercontribution to affinity under the conditions used. Nevertheless thedeletion analysis in FIGS. 1 b and 1 c clearly show that theseC-terminal residues are required for binding.

Identification of a TRAM Interaction Motif (TRIM) in E1A, Required forthe Binding of the CBP TRAM

Having identified the E1A-binding region of CBP, we were interested inidentifying the residues in E1A responsible for binding the CBP TRAM.Previous dissection of E1A has implicated residues 63-67 in binding tothis region of CBP. We therefore carried out an extensive mutagenicanalysis to establish the motif in E1A required to bind CBP. FIG. 2shows that the sequence FPESLIL can be defined as essential for thebinding to CBP (1621-1877). Mutagenesis of any two residues within thissequence (FE, PS, EL, SI or LL) abolishes binding to CBP. However,single residue substitutions in this motif are insufficient to disruptthe E1A-CBP complex, indicating that the interaction between theseproteins is reliant on a combination of residues.

Peptide competition studies confirmed these results. Peptides containingwild type or mutant E1A sequences were analysed for their ability toprevent the binding of full-length radiolabeled 12S E1A protein to aGST-CBP fusion protein (FIG. 2 b). While E1A binding was detected in theabsence of competitor peptide, increasing amounts of wild type E1Apeptide abolished the interaction. By contrast, peptides containingmutations in E67L69 (peptide Mut 1), or additionally F65L71 (peptide Mut2), failed to abolish the E1A-CBP interaction. These results demonstratethat the interaction of E1A with the CBP TRAM is via a small E1A motif.

Transcription Factors p53, E2F and TFIIB Interact with CBP Via its TRAM

The CBP TRAM is within a region of CBP (1621-1877) which is a “hot spot”for the binding of transcription factors (FIG. 1 a). We therefore askedwhether the TRAM was the target of these interactions. FIG. 2 c showsthat three transcription factors, p53, E2F and TFIIB, which bind thisregion of CBP, interact with the CBP TRAM (GST-CBP 1808-1826). Theinteraction can be competed with an E1A peptide containing the wild typeCBP binding site, but not with a mutant E1A peptide that is unable tobind CBP (Mut 2).

Identification of a Conserved TRIM Sequence in p53 and E2F

Given that E1A, p53, E2F and TFIIB recognise the same motif within CBP,it is quite likely that these proteins contain a conserved domainthrough which the interaction is mediated (a TRAM-Interaction Motif(TRIM)). Previous mutagenesis studies have defined residues in twotranscription factors, p53 and E2F, which are necessary for theinteraction with CBP. FIG. 2 d shows that these residues of p53 and E2Fshow marked similarity to the CBP binding site in E1A. A conserved motifFXE/DXXXL is present in all three proteins, which when mutatedeliminates CBP binding. Thus, the results in FIG. 2 provide a model inwhich E1A regulates the activity of certain CBP-binding proteins bypossessing a motif used by these cellular transcription factors to bindCBP.

Identification and Characterisation of a TRAM Sequence in the C-terminusof Mdm-2 that Binds TRAM-interacting Proteins

A computer-based search of other proteins which may contain a TRAMrevealed a high degree of similarity between. CBP residues 1811-1822 anda sequence at the C-terminus of the Mdm2 protein (FIG. 3 a). Theconservation overlaps precisely the TRAM sequence of CBP, as defined bythe deletion analysis in FIG. 1. In addition, the residues found to beimportant for the protein-protein interaction functions of the CBP TRAM(RK 1811/1812 and N 1814) are conserved in the Mdm2 TRAM sequence.Significantly, the Mdm2 TRAM also has the capacity to bind the sameproteins, namely E1A, p53, E2F, and TFIIB that contact the CBP TRAM(FIG. 3 b). A mutant GST-Mdm2 construct (N472A) fails to bind E1A, likeits CBP counterpart (FIG. 1 d). Thus, in the context of anothertranscriptional regulator, Mdm2, a TRAM sequence mediates interaction toa similar set of transcription factors. This binding is independent ofthe N-terminal sequences in Mdm2 that have previously been shown to bindp53 and E2F.

Mdm2 Binds to p53 Via an Mdm-2 C-terminal TRAM

The Mdm2 protein is an important regulator of p53 activity. Binding ofMdm2 through the N-terminal domain to p53 results in the repression ofp53 transactivation capacity, and also leads to the degradation of thep53 protein. Previous dissection of Mdm2 has not identified ap53-binding site at the Mdm2 C-terminus. FIG. 3 c shows that this ismost likely due to the fact that the TRAM at the C-terminus of Mdm2 ismasked in vitro by sequences N-terminal to it. Thus, the Mdm2 C-terminus421-491 is able to bind p53, but N-terminally extended peptides (223-491and 391-491) do not. Consistent with previous observations, theN-terminus of Mdm2 (1-125) also binds p53. This masking effect of theC-terminal Mdm2 TRAM was also seen for E2F (data not shown), suggestingthat it is not restricted to p53 alone.

To confirm that the TRAM of Mdm2 was able to recognise p53 in vivo, weintroduced a CMV-GST-Mdm2 (421-491) expression vector into MRC5.SV40cells. FIG. 3 d shows that purification of the GST-Mdm2 fusion protein(but not GST alone) from these cells results in the co-purification ofendogenous p53, as detected by the p53-specific DO-1 antibody. Thus theTRAM-containing C-terminus of Mdm2 represents an independent bindingsite for p53 in vivo. The relative contribution of the N- and C-terminalbinding sites for p53 in the context of full-length protein is unclearat this stage. However the in vitro data presented here raise thepossibility that the TRAM sequence of Mdm2 may be unmasked only undercertain physiological conditions.

Inspection of the masking region from 391-421 shows that a number of SQor TQ motifs are present. These sites have been shown to representpotential phosphorylation sites for DNA-dependent protein kinase(DNA-PK). This kinase is activated in response to DNA damage. Given theimportant role p53 plays in cell cycle arrest and apoptosis after DNAdamage, it is tempting to speculate that phosphorylation of SQ/TQ motifsin the Mdm2 C-terminal could result in an unmasking of the Mdm2 TRAM.

The N-terminal domain of Mdm2, and the CBP and Mdm2 TRAMs, recognise thesame region of p53. FIG. 4 a demonstrates that while a previouslydescribed p53 mutation (L14Q F19S) drastically affects the binding ofthe N-terminal domain, it does not affect the binding of the CBP or Mdm2TRAMs. This suggests that the importance of individual amino acidresidues, and therefore the contacts involved, differ in these two typesof protein interaction motif.

To determine whether or not the binding of the N-terrninal Mdm2 domainand TRAMs to p53 was mutually exclusive, we carried out a competitionassay in which a p53-Mdm2 (1-125) interaction was challenged with eithera wild type CBP TRAM-containing peptide (1806-1832) or a peptidecontaining a mutated TRAM sequence. FIG. 4 b shows that the wild-typepeptide successfully competes with the N-terminal Mdm2 domain for p53binding, while the mutant TRAM peptide's ability to compete issignificantly reduced.

TRAM Sequences Activate p53-dependent Transcription

In a recent report, disruption of the Mdm2 N-terminal-p53 interactionresulted in a striking accumulation of endogenous p53 protein,activation of p53-dependent transcription, and cell cycle arrest. Theresults presented in FIG. 4 b suggested to us that the introduction ofrRAM-containing proteins into cells containing functional p53 andfull-length Mdm2 would result in a similar effect. Transienttransfection experiments using U-2 OS cells show that the introductionof CBP sequences (1808-1852) containing a functional TRAM do indeedresult in an activation of p53-dependent transcription in adose-dependent manner (FIG. 4 c). The presence of a TRAM mutationsignificantly reduces this effect. Moreover, the activation ofp53-dependent transcription by these CBP sequences is abolished afterthe co-transfection of full-length Mdm2, which is consistent with theidea that these proteins may be competing for p53 binding in vivo.

FIG. 4 d illustrates that the ability to activate p53-dependenttranscription is not limited to the CBP TRAM, but can also be achievedusing Mdm2 C-terminal sequences containing a functional TRAM. Activationwas significantly reduced when N-terminal “masking” sequences (391-491and 223-491) were included, suggesting that masking of the Mdm2 TRAMoccurs in vivo as well as in vitro. The transfection of the N-terminalMdm2 domain (1-125), which contains p53-binding sequences but not thoseimplicated in p53 degradation, also resulted in a comparable activationof p53-dependent transcription.

The three constructs that activate p53-dependent transcription, CBP(1808-52), Mdm2 (421-91), and Mdm2 (1-125), share no obvious commonfeature other than their ability to bind p53. This strongly suggests amodel in which these proteins compete for p53 binding with theN-terminal domain of full-length endogenous Mdm2, resulting in anabrogation of Mdm2-mediated p53 degradation.

In summary, our results show that both CBP and Mdm-2 contain atranscriptional adaptor motif that recognises multiple cellularregulators, at least some of which such as E2F and p53 contain anE1A-like TRIM having the consensus FXE/DXXXL motif. Both TRAMs share anability to activate p53 function by competing with the N-terminal motifof full-length Mdm2 for p53 binding. Competition between proteins thatcontain the FXE/DXXXL motif and TRAMs may also play an important role inthe regulation of many different signal transduction pathways, andevidence obtained from the study of adenovirus E1A suggests that virusescan manipulate these protein-protein interactions to alter cell fate.This simple, motif-based interaction interface should therefore providea good target for drug-based therapeutic intervention.

The Transcriptional Regulator YY1 Contains Multiple TRIMs that Correlatewith CBP Binding and in vivo Repressioln

Yin Yang 1 (YY1) is an important regulator of numerous viral andcellular genes. Recently, we have shown that YY1 can repress AP-1mediated activation of the HPV-16 E6/ E7 promoter. Both the jun and fosfamily members that make up the AP-1 transcription factor have beenshown to use CBP as a co-factor during transcriptional activation. Wehave previously shown that YY1 can interact with CBP, while others havedemonstrated the ability of YY1 to interact with p300.

An examination of the YY1 sequence reveals the presence of threepotential TRIMs (127-133; 307-313 and 334-340). In FIG. 5 a YY1fragments used in in vitro binding studies and in vivo transcriptionrepression studies show the positions of the three TRIMs (white bars),one of which is in the N-terminal half of the protein, the others beingin the C-terminal portion as part of zinc finger structures. FIGS. 5 band 5 c demonstrate a correlation between YY1 constructs containingE1A-like TRIMs, CBP binding, and the ability to repress AP-1 activity invivo. These results suggest that a TRIM-TRAM interaction may play a rolein the modulation of gene expression by a DNA-binding factor.

In many cases YY1 serves to keep viral gene expression at low levelsuntil a particular trigger or phase in the viral life cycle when reliefof YY1 repression occurs, often mediated by other transcription factors.It may be possible therefore, to use TRIM-TRAM interactions todown-regulate specifically viral gene expression in an analogous way.

Differential Binding of TRAM Variants to TRIM-containing Proteins

The E1A, p53 and E2F TRAM interaction motifs (TRIMs) studied initiallyconsisted of the consensus FXE/DXXXL. A study of potential TRIMs fromother proteins suggests that in addition to the variation in the 7^(th)position (in which an alternative, neutral, non-polar amino acid maysubstitute for leucine), there is also significant variation within theresidues denoted by ‘X’ (see FIG. 6 a). Since the composition ofresidues within the TRIM could influence the contacts made with anygiven TRAM, we were interested to see if different TRAM variantsdemonstrated differential binding to a number of these TRIM-containingproteins.

We used the eleven alanine substitution mutants, previously created inthe context of the GST-CBP (1808-1826) fusion protein, in pull-downassays with p53, TFIIB, and YY1. It can be seen from FIG. 6 b that aswell as being affected by the mutations in R1811/K1812 (mutant 1) andN1814 (mutant 2), these TRIM-containing proteins are also affected bycertain alanine substitutions in the C-terminal part of the CBP TRAM.This is in contrast to E1A, which was not affected by these mutations.For example, the mutation K1821A (mutant 9) affects all three proteins(p53, TFIIB, and YY1), while Q1822A (mutant 10) affects p53, and YY1,but not TFIIB. In addition to this, YY1 is also affected by additionalmutations that do not affect either p53 or TFIIB.

Together, these results provide a precedent that variants of the TRAMsequence (whether naturally occurring or created by mutagenesis) canshow differential binding to different TRIM-containing proteins.Consequently, if multiple changes were introduced it might be possibleto express TRAM-containing proteins or peptides that displayed selectivespecificity that upon binding to particular TRIM-proteins could inhibittheir interaction with other TRAMs. One example where this might bedesirable would be with “high risk” HPV E6 protein where binding of asynthetic TRAM-containing peptide to E6 might be used to prevent anE6-CBP interaction to inhibit the effect that E6 has on p53.

EXAMPLE 2 Materials and Methods

Plasmid constructs. The vector used for the expression of GST-fusionproteins, unless otherwise stated, was pGEX-2TKP (a modified version ofthe Pharmacia pGEX-2TK vector containing a polylinker, Bannister &Kouzarides 1995) which was a gift from Tony Kouzarides. Also a gift fromT. Kouzarides were the plasmids GST-CBP I (residues 461-662), GST-CBP II(residues 1621-1877), GST-P/CAF, GST-E1A [1-90] (residues 1-90 of theadenovirus E1A protein), G5E1BCAT, pHK3NVP16, pHKnTCBPIVP16,pHKnCBPIIVP16 and pHKGT. This last construct contains the DNA bindingdomain of GAL4 (1-147) driven by an SV40 promoter and was used to createthe GAL4-HPV-E6 fusion proteins GAL-11E6 and GAL-16E6. Plasmids GST-11E6and GST-16E6 were created by inserting HPV-11 and HPV-16 E6 sequencesamplified by PCR into pGEX2TKP. The GST-6E6 and GSTF-18E6 constructsused to express the full-length HPV-6 and 18 E6 proteins respectively,were a kind gift from David Pim (Pim et al, 1997). The GST-E6APexpression vector was kindly provided by Elliot Androphy (Huibregtse etal, 1991). The GST-CBP constructs described in FIG. 8 (1 to 8) werecreated by cloning PCR amplified fragments into pGEX2TKP. The MBP-CBPfusion construct was created by cloning a CBP fragment (residues1808-1852) from pGEX2TKP into the vector pMALP, a modified version ofpMAL (NEB) containing a polylinker (FIG. 13) that was a gift from EdwardManser. Also provided by Dr. Manser was the in vitro transcription andmammalian expression vector pXJ-FLAG (Manser et al, 1997). The originalDNA for the HPV-16 E6 mutant L50G was a kind gift from Tadahito Kanda(Nakagawa et al, 1995) and was cloned via the BamH1 and Xho1 restrictionsites into pXJ-FLAG. All the other constructs used for in vitrotranscription reactions were cloned BamHI/HindIII into pXJ-FLAG.

Expression of recombinant bacterial fusion-proteins. GST-fusion andMBP-fusion proteins were expressed in E. coli, extracted with lysisbuffer (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 5 mM DTT, 15% glycerol, 1mg/ml lysozyme and 1 mM PMSF), and after sonication and centrifugation,stored at −70° C.

Partial-purification of CBPlp300 from HeLa Nuclear extract. HeLa nuclearextract was diluted 1:3 with 20 mM MES buffer pH 6.1, 10 mM NaF, and0.1% Triton X-100, before being passed over a 0.2 ml SP Sepharoseion-exchange column (Pharrnacia) equilibrated with the same buffer.Elution of CBP/p300 using a step gradient of increasing [KCl] wasmaximal in the 300 mM and 400 mM KCl fractions (as determined by Westernblot analysis using an anti-p300 antibody, data not shown). These twofractions were pooled and then diluted 1:7 with binding buffer (20 mMTris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 mM DTT, 20% glycerol, 01% NP40) tobring the final salt concentration down to 50 mM KCl. The partiallypurified CBP/p300 was then passed over GST-fusion protein micro-affinitycolumns as described below.

Detection of protein-protein interactions using micro-affinity columns.Bacterial lysate containing GST-fusion protein was incubated withglutathione-Sepharose beads (Pharmacia) for 30 min at 4° C. in 1×NENTbuffer (100 mM NaCl, 1 mM EDTA, 0.5% NP-40, Tris-HCl, pH 8.0). Afterspinning down and washing with 1 ml 1×NENT, the beads were loaded into ayellow Gilson pipette tip containing a glass bead (BDH, cat. no.332134Y) to create a 25 l GST micro-column. For MBP micro-columns, asimilar approach was taken in which amylose resin (NEB) was used inplace of glutathione-Sepharose beads. These columns were then used todetect interactions with in vitro translated and radiolabelled proteins,bacterially expressed fusion proteins, or partially purified nuclearCBP/p300.

For in vitro translation (IVT) proteins, expression and incorporation of³⁵S-methionine was performed using TNT kits (Promega) according to themanufacturer's recommendations. After a 1 hr incubation at 30° C., 40 μlof a 50 μl IVT reaction was diluted with 360 μl of IPD buffer (50 mMKCl, 40 mM Hepes, pH 7.5, 5 mM 2-Mercaptoethanol, 0.1% Tween-20, 0.5%milk) before being passed over the GST micro-column. After washing thecolumn twice with 200 μl wash buffer (IPD buffer containing 150 mM KCl),proteins were eluted from the column by adding 25 μl of 2×SDS loadingdye, heating to 95° C. for 5 min, chasing with 25 l water, and spinningin a micro-centrifuge. Samples were then analysed by SDS PAGE and, afterstaining and drying down the gel, proteins were detected via exposure toautoradiographic film.

In order to detect the interaction between two bacterially expressedrecombinant proteins, GST-fusion proteins were passed down MBP-fusionmicro-affinity columns or vice versa. After purification of the targetrecombinant fusion protein on glutathione Sepharose beads or amyloseresin, the proteins were eluted using recombinant binding buffer (RBB)(25 mM Hepes, pH 7.6, 50 mM KCl, 12.5 mM MgCl₂, 10% glycerol, and 0.1%NP40). In addition to the RBB, either 10 mM reduced glutathione (Sigma)(for GST-proteins) or 20 mM maltose (for MBP-fusion proteins) waspresent. After passing the recombinant target protein in RBB over themicro-affinity column and washing using RBB containing 150 mM KCl, thesamples were eluted as described above for IVT proteins and run on SDSgels. Samples were then transferred onto polyvinylidene difluoride(PVDF) membranes and GST or MBP fusion proteins detected using theappropriate antibodies by Western blot analysis (see below). A similaranalysis was performed for the partially purified nuclear CBP/p300proteins. In this case the binding buffer consisted of 20 mM Tris-HCl,pH 8.0, 50 mM KCl, 0.5 mM EDTA, 0.5 mM DTT, 20% glycerol, 01% NP40, andCBP/p300 was detected by Western analysis using antibodies specific forthese proteins.

Western blot analysis. Proteins analysed on 0.75 mm thick SDS-PAGE gelswere blotted onto PVDF membranes (NEN) overnight. The membranes wereblocked with 5% (w/v) nonfat dry milk in TBST (10 mM Tris-HCl, pH 8.0,150 mM NaCl, 0.05% Tween 20). A 1 hr incubation at room temperature withthe first monoclonal antibody was performed followed by washing withTSBT. The membrane was then incubated for 30 min with a horseradishperoxidase-coupled second antibody (1:4,000; DAKO) before washing inTSBT. Proteins were visualised with hyperfilm in the presence luminol(Amersham) for 10 to 60 s, depending on signal intensity.

Mammalian two hybrid-experiments. To study protein-protein interactionsin vivo we made use of a Gal4-VP16 CAT reporter system describedpreviously (Bannister & Kouzarides 1995). Full-length HPV 11 E6 and 16E6 sequences were fused to the DNA binding domain of GAL4 resulting inthe constructs pGAL4-11E6 and pGAL4-11E6, respectively. U2-OS cells wereco-transfected with 1 μg pGAL4-11E6 or pGAL4-11E6 and 4 μg pG5E1BCAT (aCAT reporter vector containing multiple GAL4 DNA-binding sites).Co-transfected together with these plasmids was either pHK3NVP16 (theactivation domain of VP16, residues 415-490 driven by the SV40promoter), 2 μg pHKnTCBP1VP16 (expressing CBP residues 461-662 in framewith the VP 16 activation domain), or 2 μg pHKnCBP2VP16 (expressing CBPresidues 1621-1877 in frame with the VP 16 activation domain). 48 hoursafter transfection the cells were harvested and CAT assays performed asdescribed below.

In vitro p53 degradation assay. E6-mediated degradation of p53 wasassayed using a previously described method (Nakagawa et al, 1995;Scheffer et al, 1993). Essentially, 12.5 μl of in vitro translated E6protein was mixed with 2 l of in vitro translated and ³⁵S-labelled p53in a total volume of 25 μl of assay buffer (25 mM Tris-HCl (pH7.5), 100mM NaCl, 3 mM DTT). The sample was then incubated at RT for 30, 90, or180 min. At the indicated time points the reaction was stopped by adding2×SDS loading dye and boiling for 5 min. The samples were then analysedby SDS PAGE and autoradiography.

Transfections and CAT assays. U-2OS cells were plated onto10-cm-diameter culture dishes and transfected at 50-70% confluency usinglipofectin reagent (GIBCO-BRL). Chloramphenicol acetyl transferase (CAT)assays have been described elsewhere (O'Conner & Bernard, 1995) and thedata presented represent between three and eight experiments using atleast two independent DNA preparations.

Results

The HPV-16 E6 protein interacts with full-length nuclear CBP/p300. Inorder to determine whether or not the papillomavirus E6 protein couldinteract with CBP/p300, we partially purified these transcriptionalcoactivators from HeLa nuclear extract (see Materials and Methods) andthen passed the fraction enriched for CBP/p300 over an E6 affinitycolumn. Western analysis using the monoclonal antibodies p300 Ab-1 (FIG.7A), and NM11 (data not shown), detected a specific interaction betweenCBP/p300 and GST-16E6. No interaction was detected for the control GSTcolumn, even though a greater amount of protein was used. Also shown inFIG. 7A is the interaction between full-length nuclear CBP/p300 andGST-P/CAF, and GST-YY1. These data provide the first evidence that apapillomavirus oncoprotein can associate with the transcriptionalco-activator CBP/p300.

Both Ad E1A and SV40 TAg bind the CBP II domain of CBPI(residues1621-1877) which represents a hot spot for transcription factorinteractions. We tested whether or not HPV-16 E6 was also able to bindto this region of CBP using a micro-affinity column (described inMaterials and Methods) containing GST-CBP (1621-1877). In FIG. 7B it capbe seen that in vitro translated and radiolabelled HPV-16 E6 does indeedbind to the GST-CBP II domain, but not to GST or to GST-CBP I (461-662),another region of CBP that binds multiple cellular transcriptionfactors. Furthermore, our unpublished results suggest that unlike E1A,HPV-16 E6 does not bind to multiple regions of CBP/p300 but is limitedto the CBP II domain.

In order to gain an insight into the relative strength of the E6-CBP IIassociation, we compared this protein-protein interaction with twopreviously described interactions, namely that of E1A and the CBP IIdomain, and the binding of HPV E6 to the cellular factor E6AP. As can beseen from the results presented in FIG. 7C, the association of HPV-16 E6with the CBP II domain is of a similar strength to those seen with thetwo previously documented interactions. Nevertheless, it should be notedthat while HPV-16 E6 and Ad E1A bind the CBP II domain at a comparablelevel, E1A binds full-length nuclear CBP/p300 with a much higheraffinity. This is most likely due to the fact that E1A can bind multiplesites on CBP/p300 in addition to the CBP II domain.

FIG. 7D demonstrates that the interaction between HPV-16 E6 and the CBPII domain can occur directly, since binding can be detected using onlypurified, recombinant proteins. The interaction of GST-16E6 with anMBP-CBP affinity column was detected by western analysis using specificGST antibodies, while in the reciprocal experiment MBP-CBP binding to aGST-E6 column was detected using an anti-MBP antibody.

Together, these results provide evidence that the human papillomavirustype-16 E6 protein can associate with full-length nuclear CBP/p300 viathe CBP II domain in an interaction that is most likely direct.Interestingly, we have not been able to detect the interaction of HPV-16E6 with full-length in vitro translated CBP or p300, suggesting thatpost-translational modification of CBP/p300 may be required for theinteraction with HPV-16 E6.

Characterisation of the HPV-16 E6-CBP interaction. In order to determinethe E6 binding site within the CBP II domain, we utilised a number ofGST-CBP constructs in micro-affinity column assays with in vitrotranslated and radiolabeled full-length HPV-16 E6 protein (see FIG. 8).We were able to identify a 19 amino acid region of CBP (1808-1826) thatwas capable of binding full-length E6 (lane 7). Deletion into thissequence abolished binding to the E6 protein (lane 8).

Interestingly, these CBP residues, presented in FIG. 8, are identical tothose we recently characterised as the binding site for the Ad E1Aprotein, as well as numerous cellular transcription factors includingp53. Indeed, FIG. 8 also shows the ability of a wild type E1A peptidethat can bind the 19 amino acid CBP sequence to inhibit the HPV-16E6-CBP interaction. A mutant E1A peptide that is unable to bind CBPfails to inhibit the E6-CBP interaction. It can be seen that thissequence is virtually identical in both CBP and p300, with only oneconservative change in the 19 amino acid segment. This sequence is alsohighly conserved in other CBP/p300, and may represent the majortranscription factor-binding site within the CBP II domain species.

A similar analysis of the CBP-binding site-within HPV-16 E6 was alsoperformed. As can be seen from FIG. 9, removal of the C-terminalresidues 148-151 that have been implicated in the binding of anotherE6-interacting protein, hDLG, had no effect on CBP banding. Dissectionof the HPV-16 E6 protein into N-terminal (1-84) and C-terminal (85-151)halves demonstrated that while the N-terminus of E6 does not bind CBP,the C-terminal half of the protein maintained the ability to bind CBP,as did a smaller C-terminal region (amino acids 100-147).

In the context of a GST-fusion protein (E6 amino acids 100-142), thecystein residues (C103, C139 and C140) could be substituted with glycineresidues without affecting the ability to bind CBP (our unpublishedresults). This suggests that specific sequences within the second zincfinger of E6 are involved in the interaction with CBP and that, at leastin this context, an intact zinc finger structure is not necessary.However, we have not tested these mutations in the context offull-length E6 alone, and cannot therefore rule out that an intact zincfinger structure is necessary to present the E6 residues contacting CBPunder these conditions. We are currently attempting to define moreprecisely the HPV-16 E6 residues involved in the interaction with CBP.

“High-risk” but not “low-risk” HPV E6 proteins bind CBP/p300. It hasbeen suggested that functional differences between the E6 and E7proteins of different HPVs is a cardinal factor in the ability of theseviruses to transform cells and is also reflected by their classificationas either high-risk or low-risk. In the case of other DNA tumour virusproteins, such as the Ad E1A protein and the SV40 TAg, interaction withthe transcriptional coactivators CBP/p300 has been shown to beabsolutely required for their transforming capabilities. If CBP/p300 isconsidered an important target in the transformation processes of otherDNA tumour viruses, we postulated that an ability to target CBP/p300might also be an important factor in distinguishing high-risk E6proteins from low-risk proteins. Consequently, we investigated theability of another high-risk E6 protein (from HPV-18) to bind CBP, andcompared this, along with HPV-16 E6, to two low-risk E6 proteins fromHPV-6 and HPV-11.

FIG. 10A does in fact show that only the GST-E6 proteins of thehigh-risk types (HPV-16 and HPV-18) can bind to in vitro translated CBPII, while those of the low-risk types (HPV-6 and HPV-11) fail to bindCBP above the background level. This observation is reproducible, as canbe seen from the inability of in vitro translated HPV-11 E6 protein tobind to a GST-CBP affinity column.

This difference in CBP binding is also observed in vivo, as demonstratedby the mammalian two-hybrid assay presented in FIG. 10B. Transientco-transfection experiments were performed using U2-OS cells in which aCAT reporter construct, driven by multiple GAL4 binding sites(G5E1BCAT), was introduced along with either an expression vector forfull-length HPV-11 E6 fused to the DNA binding domain of GAL4(GAL-11E6), or a similar construct containing HPV-16 sequences(GAL-16E6). Activation of transcription was then determined for thosecells containing these two plasmids in conjunction with either theexpression vector for the VP16 activation domain alone, VP16 fused tothe CBP I domain, or VP16 fused to the CBP II domain. The level of CATactivity obtained with cells co-transfected with the VP16 activationdomain was set at one and the CAT activity of cells receiving either CBPI-VP16, or CBP II-VP16 was then compared to this.

As can be seen from the results in FIG. 10B, cells containing GAL-16E6could be activated by CBP II-VP 16, while those containing the GAL-11E6expression vector could not. This effect was specific for the 16E6-CBPII interaction, since VP16 sequences fused to the CBP I domain failed toactivate GAL-16E6. Taken together, these results strongly suggest thatthere are functional differences between high-risk and low-risk proteinswith respect to their ability to bind CBP/p300 both in vitro and invivo.

The down-regulation of p53 transcriptional activity by HPV-16 E6correlates with CBP binding. One of the main functions proposed forhigh-risk HPV E6 proteins is the targeting of p53 in order to suppressapoptosis of the host cell. In the last few years, many lines ofevidence have suggested that one way in which this might be achieved isby stimulating the degradation of p53 through the ubiquitinationpathway. Evidence has been provided both in vitro and in vivo that thisactivity is dependent upon the interaction of E6 with a cellular factortermed E6AP which then acts as a ubiquitin ligase. The ability of E6proteins to interact with E6AP has been shown to be limited to those ofhigh-risk HPV types. It has also been reported previously that high-riskbut not low-risk E6 proteins are able to down-regulate p53transcriptional activity. One explanation for these observations is thatdown-regulation of p53-dependent transcription results from theE6AP-dependent degradation of p53.

Recently, it was also shown that p53-dependent transcription can beactivated by CBP/p300 and that this activation can be abrogated by wildtype E1A, but not a CBP-binding deficient mutant of E1A. The resultspresented here have demonstrated that like E1A, high-risk HPV E6proteins can also target CBP/p300. We therefore asked the questionwhether or not the down-regulation of p53 transcriptional activity byhigh-risk E6 proteins could be achieved through the binding of CBP/p300in a similar fashion to E1A.

In order to answer this question, we required a high-risk E6 mutant thatwas deficient in targeting p53 for degradation through the E6AP pathwayyet was still capable of binding CBP/p300. We analysed a number ofexisting HPV-16 E6 mutants before finding one with the desiredproperties. The 16E6 mutant L50G contains a point mutation in the firstzinc finger of the E6 protein and has previously been shown to bep53-degradation-deficient. FIG. 11A demonstrates that this mutant isstill able to interact with CBP in binding assays. However, when wetested this mutant for its ability to bind E6AP in a similar assay, wefound that it was deficient in this capacity (see FIG. 11B).Furthermore, FIG. 11C confirms that this mutant, like the low-riskHPV-11 E6 protein, is unable to degrade p53 using a standard degradationassay previously described.

We next carried out a series of experiments in which we assessed theability of the 16E6 L50G mutant to down-regulate p53-dependenttranscription. U2-OS cells were transfected with the p53-responsive CATreporter PG13CAT or the control vector MG15CAT. Co-transfected withPG13CAT were various expression plasmids coding for E6 proteins or 12SE1A. It can be seen from FIG. 12A that the PG13CAT is stimulated byendogenous p53 in U2-OS cells in a manner dependent upon intactp53-binding sites. The level of transcriptional activity obtained withPG13CAT is not affected by the introduction of an expression vectorcontaining full-length HPV-11 E6 sequences. By contrast, the expressionof wild type 16E6 protein results in a significant reduction inp53-dependent transcription. Consistent with our earlier analysis of theCBP-binding domain within 16E6, the N-terminal 84 amino acids that donot bind CBP, fail to repress p53 activity, while the C-terminal half of16E6 that contains the CBP-binding domain can repress p53-dependenttranscription, albeit slightly less efficiently than the full-lengthprotein. Also shown for comparison is the level of repression of p53activity obtained upon the introduction of the adenovirus 12S E1Aprotein. Significantly, the 16E6 L50G mutant results in a similar levelof transcriptional repression as the wild type HPV-16 E6 protein. Thus,in this respect the 16E6 L50G mutant does not behave like a low-riskprotein, but rather like wild type 16E6. These data provide evidencethat are consistent with the idea that by targeting CBP/p300 a high-riskE6 protein can repress p53 transcriptional activity. Furthermore, theuse of the 16E6 L50G mutant suggests this ability is independent ofE6AP-mediated degradation.

We wished to provide further evidence that the repression of p53transcriptional activity by 16E6, 16E6 L50G, and E1A was due to thetargeting of CBP/p300. Therefore, we over-expressed full-length CBP in asimilar set of transfection experiments and asked whether or not theobserved superactivation of p53-dependent transcription could beabrogated by these proteins. FIG. 12B demonstrates that theco-transfection of full-length CBP into U2-OS cells stimulatesp53-dependent transcription by approximately 7-fold. Like E1A, both wildtype 16E6 and the 16E6 L50G mutant abolish this CBP-inducedsuperactivation of p53-dependent transcription. This is in contrast toHPV-11 E6 which is severely abrogated in this capacity.

In summary, the results presented in FIGS. 11 and 12 provide evidencethat high-risk E6 proteins possess an additional and, up until now,undiscovered mechanism by which to down-regulate p53 activity. That is,by targeting CBP/p300 high-risk E6 proteins can abrogate p53-dependenttranscription in a fashion analogous to adenovirus E1A.

References

-   Bannister, A. J. and T. Kouzarides., 1996, CPB-induced stimulation    of c-Fos activity is abrogated by E1A. EMBO. J. 14: 4758-4762.-   Huibregtse, J. M., M. Scheffner, and P. M. Howley, 1991, A cellular    protein mediates association of p53 with the E6 oncoprotein of human    papillomavirus types 16 or 18. EMBO J. 10:4129-4135.-   Manser, E., H. Y. Huang, T. H. Loo, X. Q. Chen, J. M. Dong, T.    Leung, and L. Lim., 1997, Expression of constitutively active    alpha-PAK reveals effects of the kinase on actin and focal    complexes. Mol. Cell. Biol. 17:1129-1143.-   Nakagawa, S., S. Watanabe, H. Yoshikawa, Y. Taketani, K. Yoshiike,    and T. Kanda, 1995, Mutational analysis of human papillomavirus type    16 E6 protein; transforming function for human cells and degradation    of p53 in vitro., Virology 212, 535-542.-   O'Conner, M. and H. U. Bernard, 1995, Oct-1 activates the    epithelial-specific enhancer of human papillomarvirus type 16 via a    synergistic interaction with NFI at a conserved composite regulatory    element., Virology 207, 77-88.-   O'C.onnor, M. J., S. H. Tan, C. H. Tan, and H. U. Bernard. 1996. YY1    represses human papillomavirus type 16 transcription by quenching    AP-1 activity. J. Virol. 70: 6529-6539.-   Pim, D., P. Massimi, and L. Banks, 1997, Alternatively spliced    HPV-18 E6* protein inhibits E6 mediated degradation of p53 and    suppresses transformed cell growth. Oncogene 15, 257-264.-   Scheffner, M., J. M. Huibregtse, R. D. Viersta, and P. M. Howley,    1993, The HPV-16 E6 and E6-AP complex functions as a    ubiquitin-protein ligase in the ubiquitination of p53. Cell 75,    496-505.-   Smith, D. B., and Johnson, K. S. 1988. Single-step purification of    polypeptides expressed in Escherichia coli as fusions with    glutathione S-transferase. Gene 67: 31-40.-   Xiao, J. H., I. Davidson, H. Matthes, J. -M. Gamier, and P.    Chambon. 1991. Cloning, expression and transcriptional properties of    the human enhancer factor TEF-1. Cell 65: 551-568.-   Unckell, F., Streeck, R., and Sapp, M. 1997. J. Virol. 71(4):    2934-2939.

1. A method for determining whether a compound is capable of inhibitingan interaction between a first polypeptide and a second polypeptide saidmethod comprising: (a) (i) incubating said first polypeptide with saidsecond polypeptide in vitro under conditions which allow the firstpolypeptide to bind to the second polypeptide to form a complex; andbringing the complex thus formed into contact with a candidate compound;or (ii) incubating said first polypeptide with said second polypeptidein vitro in the presence of a candidate compound under conditions whichwould allow the first polypeptide to bind to the second polypeptide inthe absence of the candidate compound; and (b) determining if saidcandidate compound inhibits binding of the first polypeptide to thesecond polypeptide; wherein said first polypeptide comprises aTranscriptional Adaptor Motif (TRAM) sequence of any one of thesequences shown in SEQ ID NO: 3 to 9 and said second polypeptide is ahuman papillomavirus (HPV) polypeptide E6 of HPV-16 or HPV-18 comprisinga sequence which binds to a said TRAM sequence.
 2. The method accordingto claim 1 wherein said first polypeptide is a polypeptide found ineukaryotic cells.
 3. The method according to claim 2 wherein saideukaryotic polypeptide is selected from transcription factors and cellcycle regulatory proteins.
 4. The method according to claim 2 whereinsaid eukaryotic polypeptide is selected from mdm2 comprising a sequenceof SEQ ID NO:6 or 7, CREB binding protein (CBP) comprising a sequence ofSEQ ID NO:3, 4 or 5, and p300 comprising a sequence of SEQ ID NO: 8 or9.
 5. The method according to claim 1 wherein the first polypeptide isCREB binding protein (CBP) comprising a sequence of SEQ ID NO:3, 4 or 5.6. The method according to claim 1 wherein the sequence which binds saidTRAM sequence is located within the second zinc finger of HPV-16 or -18E6 protein.
 7. The method according to claim 1 wherein said firstpolypeptide is p300 comprising a sequence of SEQ ID NO:8 or
 9. 8. Themethod according to claim 1 wherein said second polypeptide comprisesthe sequence of amino acids 100 to 147 of SEQ ID NO:
 18. 9. The methodaccording to claim 1 wherein the first polypeptide is mdm2 comprising asequence of SEQ ID NO:6 or SEQ ID NO:7.